PROJECT SUMMARY Recapitulation of normal lung function following a severe acute injury implies an inherent capability of lung to regenerate but the sources and relative regenerative capacities of lung epithelial stem/progenitor cells remain unclear. Although virtually all differentiated epithelial lineages can re-enter the cell cycle, increasing evidence indicates that subpopulations of distal airway cells may be particularly robust in their capacity to expand, migrate, and reconstitute alveolar barriers. Recent studies from our lab have uncovered Sox2pos/p63neg epithelial progenitors (EPs) with regenerative potential and ability to differentiate towards alveolar fate. Further, numerous other studies have described a fraction of Scgb1a1-labeled cells to also have in vitro and in vivo regenerative capacity. However, their exact identities, location, and regulation remains unclear. To further define the EPs, we performed large scale single cell sequencing of flow-sorted lung epithelial cells enriched in EPs, which identified a quiescent EP population. These cells, with a transcriptomic similarity to embryonic Sox9pos lung bud cells, have increased expression of interferon (IFN)-regulated genes. Interestingly, these cells also express a number of genes associated with cell cycle and key cyclin inhibitors Cdkn1a (p21) and Cdkn1c (p57), suggestive of their ability to proliferate under favorable conditions. Indeed, IFNpos cells represent the regenerative fraction of the airway epithelium (including that of Scgb1a1-labeled cells) in various in vitro conditions tested. Importantly, these cells also have the ability to differentiate towards either basal (marked by Cytokeratin 5 expression) or alveolar (marked by surfactant protein C expression) cells. Thus, my preliminary data have identified the regenerative fraction of the airway epithelium. However, their regulation in vivo and their ability to functionally contribute to lung epithelial regeneration post injury remain unknown. To this end, we hypothesize that low levels of constitutive IFN signaling regulates and maintains a pool of rare airway EPs that mobilize to regenerate alveolar epithelium following injury. We will test this hypothesis in following two aims: 1) To determine the role of IFN signaling in maintenance of quiescent p63neg EPs in uninjured mouse lung. 2) Determine the role of IFN signaling in in vivo responses of p63neg EPs following major injury. We will leverage mouse models to abrogate active IFN signaling in injured mice to study its role in maintenance of EPs. Likewise, conditional airway epithelial deletion of Stat1 followed by injury will determine the role of IFN/Stat1 signaling in injury response. Finally, exogenously expanded EPs will be transplanted in injured lungs and functional recovery will be measured by oxygenation and lung function. This project has a significant potential to clarify the signaling pathways regulating lung epithelial progenitor cell response during quiescence and determine the potential of these epithelial progenitors in aiding lung regeneration following an injury. Further, the novel studies involving transplantation of exogenously expanded progenitors will lay foundation for their overall regenerative potential as a therapeutic agent for future cell based therapies.